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Environmental modulation and metabolism of cyanobacterial β-N-methylamino-L-alanine
Expanded Title:Background and motivation for the research β-N-methylamino-L-alanine (BMAA) is a neurotoxic amino acid produced by most, if not all, cyanobacteria (Cox et al., 2005; Esterhuizen & Downing, 2008) and linked to amyotrophic lateral sclersosis/Parkinsonism dementia complex (ALS/PDC) (Cox et al., 2003). Cyanobacteria are ubiquitous and cosmopolitan. They occur in freshwater impoundments in very high densities due primarily to eutrophication and this poses a potential exposure risk (Codd et al., 1999). Evidence exists for various mechanisms of exposure to BMAA from cyanobacterial biomass, including ingestion of contaminated water, aquatic organisms and irrigated vegetables, and via inhalation of aerosolized toxin or toxin-containing cells. However, the reported BMAA content of cyanobacteria has varied substantially from none detected, to mg/g dry weight. Typically BMAA content ranges from a few μg/g dry weight to several hundred μg/g dry weight, but nothing is known about the cause of this reported variation in the BMAA content of cyanobacteria, or about regulation of BMAA content or about biosynthesis of BMAA in cyanobacteria. This lack of understanding of the causes of variation and possible environmental modulators makes management of risk exposure almost impossible. Project objectives The purpose of this project was to determine causes of variation in BMAA content in cyanobacteria while confirming the cyanobacterial origin of BMAA and elucidating possible biosynthetic mechanisms of BMAA in cyanobacteria. The specific project aims were as follows: o To determine the effect of variation in nutrients on the production of BMAA with an emphasis on the effect of nitrogen on BMAA content o To investigate possible mechanisms of biosynthesis and metabolism of BMAA in cyanobacteria o To investigate the possibility of estimating BMAA content based on a more easily measurable metabolite. Additionally, and due to no clear alternative metabolite being identified that was easier to quantify, an assessment of a commercial BMAA ELISA kit, that became available during the course of this project, was added as an aim. Summary of the major results and conclusions BMAA was shown to be a product of cyanobacterial metabolism. This was achieved by inducing production of BMAA using a stable isotope of nitrogen in the form of ammonia as a supplied nutrient, and observing labelled BMAA production as a consequence. BMAA is environmentally modulated. Transient increases were observed as a result of combined nitrogen deprivation and increased light intensity or medium phosphorous concentration. However, light and phosphorous responses were probably a function of alteration of cellular nitrogen status, as these variables had no effect under nitrogen-replete conditions. Medium combined nitrogen availability resulted in a rapid removal of free cellular BMAA, with ammonia having an almost immediate effect whereas nitrate had a somewhat delayed effect. These data suggest a role for BMAA in nitrogen stress response in cyanobacteria and offer a basis for environmental studies on BMAA content as a function of physicochemical parameters in surface waters. Furthermore, the immediate role of ammonia in the appearance and disappearance of BMAA offers a starting point for investigating BMAA biosynthesis in cyanobacteria. BMAA is therefore a cyanobacterial metabolite produced under nitrogen deprivation. In order to try and understand the biosynthesis of BMAA, feeding experiments with labelled nitrogen were conducted. Isotopically labelled BMAA was synthesised and this and unlabelled BMAA and other potential metabolites were used in feeding experiments under conditions that would induce either BMAA production or BMAA removal. BMAA is not produced concurrently with proteinogenic amino acid anabolism. BMAA is produced at the onset of nitrogen limitation but is not produced as a catabolic byproduct of serine protease degradation of cellular components, or directly as a result of cyanophycin or phycobiliprotein catabolism. The transient increase of free cellular BMAA observed in cyanobacteria as a function of decreased cellular nitrogen status suggests a regulatory or metabolic intermediate function for BMAA in cyanobacteria. Published hypothesized biosynthetic mechanisms were shown to be incorrect. BMAA biosynthesis does appear to be enhanced by feeding on amino acids that are transamination products of glutamate, suggesting glutamate as a potential precursor for BMAA. Additionally, BMAA appears to serve as an amino group donor in transamination reactions with either 2-oxoglutarate, oxaloacetate or pyruvate; the resulting BMAA keto acid (2-oxo-3(aminomethyl)propanoic acid) is a substrate for either amination or transamination and/or carbamanation and possibly cyclisation, to form a precursor pool from whence BMAA may rapidly be produced when required by the cells. The rapid transfer of the BMAA alpha-amino group to glutamate suggests that BMAA may serve as an amino donor in the reaction catalysed by one of the 2-oxoglutarate transaminases. The ready reversibility of transaminase reactions suggests a keto acid precursor for BMAA with glutamate as the amino group donor. However, this keto acid has yet to be detected, suggesting that its presence is transient and it is itself a product, either an isomer or otherwise altered precursor. The existence of aminated alpha and beta-carbamate derivatives of the BMAA, and the possibility of aminated forms of these molecules, presents several possible precursor molecule options that become available on nitrogen deprivation, such as the possible decarbamation of a deaminated beta-carbamate of the keto-acid of BMAA with subsequent transamination to yield BMAA. Results from these experiments therefore suggest that BMAA is rapidly produced from another, as yet unidentified, molecule in the cell, possibly by deamination and decarbamation of aminated BMAA carbamates possibly following de-cyclisation. This is supported by the rapid removal of BMAA on addition of ammonia to the medium and the existence of both the alpha-aminated and beta-aminated ethyl-carbamates of BMAA. Additionally, BMAA is subject to transamination reactions as determined by transfer of alpha 15N to the amino acid pool. Although the corresponding keto acid was not observed, this molecule may itself be subject to modification within the cell, making detection difficult. The nature of any such modification remains unknown. BMAA remains a molecule of concern. These data show that BMAA appears to have a primary regulatory or metabolic function that explains its almost universal presence within the cyanobacteria. We suggest that the absence of BMAA in wild strains analyzed is probably due to inappropriate environmental or culture conditions and that the absence in certain culture collection strains can be explained by loss of this function due to many generations under optimal growth conditions. The flux between BMAA and its metabolic precursors and products is rapid and environmentally modulated and this needs to be considered when monitoring programs are established. We have no knowledge of the potential toxicity of the metabolites of BMAA or of whether these metabolites are subject to metabolic conversion to BMAA upon ingestion. Recommendations Our understanding of the transient nature of BMAA in cyanobacteria indicates a requirement for frequent monitoring of cyanobacteria in drinking water sources where elevated chlorophyll content is detected. In the absence of conclusive data on the possible exposure routes and experimental validation of the ALS/PDC theory, it seems prudent to take measures, such as the placement of warning signs, to prevent any exposure. These data offer the first indication of the possibility of developing alert levels for BMAA based on commonly measured physicochemical parameters. The complex nature of the apparent nitrogen:carbon ratio regulation of BMAA production indicates the necessity for a long-term monitoring program wherein all relevant physicochemical parameters are measured in conjunction with BMAA so as to develop an applicable, environmental model for BMAA risk so as to inform an alert level guideline and better manage exposure risk. The insights gained into the possible regulatory function of BMAA in cyanobacteria require an urgent follow-up study to confirm the function of this molecule and thereby supply a fundamental physiological basis for any environmental parameter-based alert level framework. We therefore recommend: a) that research into the role of BMAA as a response regulator be completed so as to support environmentally- based models of BMAA presence, and b) that a long-term monitoring project be initiated to collect adequate data to support or refute the laboratory findings on physicochemical parameter-based prediction of BMAA levels in cyanobacterial blooms. The current findings, together with the recommended work, will provide a sound basis for an alert level framework for the analysis of BMAA in recreational and potable water resources.  
Date Published:01/07/2014
Document Type:Research Report
Document Subjects:Wastewater Management - Domestic, Wastewater Management - Sewers
Document Keywords:Municipality, Water Quality
Document Format:Report
Document File Type:pdf
Research Report Type:Standard
WRC Report No:2065/1/14
ISBN No:978-1-4312-0567-7
Authors:Downing TG; Downing S
Project No:K5/2065
Originator:WRC
Organizations:Nelson Mandela Metropolitan University
Document Size:607 KB
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